1. Field of the Invention
The present invention relates to a method of improving the plasma (blood) kinetics of anti-glypican 3 antibodies, a pharmaceutical composition comprising an anti-glypican 3 antibody that has improved plasma kinetics as an active ingredient, and a method of preparing the same.
2. Description of the Related Art
Antibodies are stable in the plasma and exhibit few side effects and for these reasons their use as drugs has been receiving attention. Among the several antibody isotypes, a large number of IgG isotype therapeutic antibodies are on the market and a large number of therapeutic antibodies are also currently under development (Janice M. Reichert, Clark J. Rosensweig, Laura B. Faden, and Matthew C. Dewitz, Monoclonal antibody successes in the clinic, Nature Biotechnology (2005) 23, 1073-8; Pavlou A. K. and Belsey M. J., The therapeutic antibodies market to 2008; Eur. J. Pharm. Biopharm. (2005) 59(3), 389-96; and Janice M. Reichert and Viia E. Valge-Archer, Development trends for monoclonal antibody cancer therapeutics, Nat. Rev. Drug Disc. (2007) 6, 349-356). Anti-glypican 3 antibodies are known to exhibit antitumor activity by exercising cytotoxicity against, for example, liver cancer cells and lung cancer cells (WO 2003/000883). Antibody-drug conjugates comprising an anti-glypican 3 antibody attached to a cytotoxic substance are also known to exhibit antitumor activity against liver cancer, ovarian cancer, melanoma, and so forth (Albina Nesterova, Paul J. Carter, and Leia M. Smith, Glypican 3 as a Novel Target for an Antibody-Drug Conjugate, AACR Abstract No. 656 (2007), Los Angeles, Calif., April, 4-18).
In addition, technologies to enhance the effector functions are being developed for producing second-generation therapeutic antibodies. For example, it is known that the antibody-dependent cellular cytotoxicity (ADCC) activity and the complement-dependent cytotoxicity (CDC) activity are enhanced by an amino acid substitution in which the amino acids constituting the Fc region of IgG isotype antibodies (referred to as IgG antibodies) are replaced by different amino acids (Kim S. J., Park Y., and Hong H. J., Antibody engineering for the development of therapeutic antibodies, Mol. Cells (2005) 20(1), 17-29). When an anti-glypican 3 antibody is produced in fucose transporter-deleted CHO cells, fucose is not attached to the branched sugar chains attached to the anti-glypican 3 antibody. Such an anti-glypican 3 antibody has a significantly higher ADCC activity than the anti-glypican 3 antibody that contains fucose in the branched-chain of the sugar chain, and is thought to exhibit a greater antitumor activity as a therapeutic antibody (WO 2006/067913).
In addition to such technologies for enhancing the effector functions, other technologies are also known in which the plasma half-life of an antibody is increased or decreased by amino acid substitution on the amino acids constituting the Fc region of the antibody (Hinton P. R., Xiong J. M., Johlfs M. G., Tang M. T., Keller S., and Tsurushita N., An engineered human IgG1 antibody with longer serum half-life, J. Immunol. (2006) 176(1), 346-56; and Ghetie V., Popov S., Borvak J., Radu C., Matesoi D., Medesan C., Ober R. J., and Ward E. S., Increasing the serum persistence of an IgG fragment by random mutagenesis, Nat. Biotechnol. (1997) 15(7), 637-40). If a technology that prolongs the plasma half-life of antibodies is applied to therapeutic antibodies, it is expected that the dose of the administered therapeutic antibody is reduced and its interval of administration is extended, which will enable the provision of less expensive therapeutic antibodies with a high convenience factor.
In specific terms, the plasma half-life can be extended by substituting an amino acid of the Fc region of an IgG antibody with another amino acid resulting in improving the IgG antibody's affinity for the neonatal Fc receptor, which is known to be a salvage receptor for the IgG antibody. In addition, it is also known that the plasma half-life is increased by shuffling the individual domains (CH1, CH2, CH3) constituting the constant region of the antibody (Zuckier L. S., Chang C. J., Scharff M. D., and Morrison S. L., Chimeric human-mouse IgG antibodies with shuffled constant region exons demonstrate that multiple domains contribute to in vivo half-life, Cancer Res. (1998) 58(17), 3905-8). However, since the amino acid sequence of the constant region of the IgG antibody is conserved in humans, an antibody having an artificial amino acid substitution as described above in the amino acids constituting the constant region may cause side effects by exhibiting immunogenicity in the human body. It is therefore preferred that only a small number of amino acids be substituted.
Technologies involving amino acid substitution in the variable region (also referred to as V region) of IgG antibodies reported to date include humanization technology (Tsurushita N., Hinton P. R., and Kumar S., Design of humanized antibodies: from anti-Tac to Zenapax, Methods (2005) 36(1), 69-83), affinity maturation where amino acids in the complementarity-determining region (CDR) is substituted in order to increase the binding activity (Rajpal A., Beyaz N., Haber L., Cappuccilli G., Yee H., Bhatt R. R., Takeuchi T., Lerner R. A., and Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries, Proc. Natl. Acad. Sci. USA (2005) 102(24), 8466-71) and amino acid substitution in the amino acids constituting the framework region (FR) for improving the physicochemical stability (Ewert S., Honegger A., and Pluckthun A., Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering, Methods (2004) 34(2), 184-99). Unlike the case with amino acid substitution in the constant region (also referred to as C region), amino acid substitution in the variable region is generally used for improving the characteristics (e.g., stability) and enhancing the function (e.g., antigen binding activity) of antibodies. Since the amino acid sequence constituting the CDR of humanized antibodies is derived from the amino acid sequence of a nonhuman animal species, the risk of generating immunogenicity by introducing an artificial amino acid substitution in this sequence is thought to be lower than amino acid substitutions in a sequence in other regions. Moreover, with regard to an artificial amino acid substitution in the amino acid sequence constituting the FR of humanized antibodies, it is thought that such a substitution poses little risk of generating immunogenicity if the FR amino acid sequence obtained as a consequence of substitution is the same as any of the plurality of human antibody FR amino acid sequences that are published in, for example, the Kabat database (ftp.ebi.ac.uk/pub/databases/kabat/), the IMGT database (imgt.cines.fr/), and so forth. Furthermore, the immunogenicity can be reduced by reselecting a human antibody sequence that is very similar to the FR amino acid sequence obtained as a consequence of substitution, from the plurality of human antibody FR amino sequences that are published in the Kabat database, the IMGT database, and so forth (WO 1999/018212).
In contrast, the only methods known for improving the plasma half-life of IgG antibodies are, as described above, amino acid substitution of amino acids constituting the Fc region, which is a part of the constant region, and no methods have been reported to date that bring about an improvement in the plasma half-life of IgG antibodies by amino acid substitution of the amino acids constituting the variable region, which is believed to carry little risk of invoking immunogenicity. The reason for this is, in part, that the plasma half-life of IgG antibodies are believed to largely depend on antigen-dependent depletion and binding to the neonatal Fc receptor, a salvage receptor for IgG antibodies (Lobo E. D., Hansen R. J., and Balthasar J. P., Antibody pharmacokinetics and pharmacodynamics, J. Pharm. Sci. (2004) 93(11), 2645-68), and that the functions and properties of the variable region may not have a significant influence on the plasma half-life.
It has also been reported that the isoelectric point (pI) of IgG antibody is lowered by anionization of IgG antibody by succinylation (Yamasaki Y., Sumimoto K., Nishikawa M., Yamashita F., Yamaoka K., Hashida M., and Takakura Y., Pharmacokinetic analysis of in vivo disposition of succinylated proteins targeted to liver nonparenchymal cells via scavenger receptors: importance of molecular size and negative charge density for in vivo recognition by receptors, Pharmacol. Exp. Ther. (2002) 301(2), 467-77); and that the pI of IgG antibody is raised by cationization of the IgG antibody by modification with polyamine (Poduslo J. F. and Curran G. L., Polyamine modification increases the permeability of proteins at the blood-nerve and blood-brain barriers, Neurochem. (1996) 66(4), 1599-609). However, in both cases there was no increase in the plasma half-life of the modified IgG antibody, but rather the plasma half-life was decreased. Thus, an increase in the plasma half-life of IgG antibodies cannot be realized by modification of the pI of the IgG antibody by the above-described chemical modification of the IgG antibody.